The role of individual cell types in hepatic hypoxia-reoxygenation (reperfusion) injury has not been completely defined. We therefore examined the effects of hypoxia and hypoxia-reoxygenation on the viability of rat hepatocytes, Kupffer cells, and sinusoidal endothelial cells (SECs) in primary culture and whether direct exposure to hypoxia followed by reoxygenation activated Kupffer cells. Cultures of hepatocytes (purity > 99%), Kupffer cells (97%), and endothelial cells (> 93%) were established as single-cell types and as cocultures. Hypoxia was achieved by culturing cells under 95% N2/5% CO2, and cell viability was estimated by lactate dehydrogenase (LDH) leakage and Trypan blue exclusion. Kupffer cells and endothelial cells were more resistant to hypoxia than hepatocytes. Following 4-8 hours of hypoxia, reoxygenation accentuated cell death in endothelial cells. In contrast, reoxygenation did not accentuate cell death in hepatocytes or in resting Kupffer cells. The activation of Kupffer cells by the addition of lipopolysaccharide failed to alter their response to hypoxia-reoxygenation. The addition of phorbol myristate acetate to Kupffer cells stimulated the production of superoxide as expected, and the medium from these activated cells augmented the cellular injury of hypoxic hepatocytes. In contrast, hypoxia-reoxygenation did not stimulate Kupffer cells to produce superoxide or other hepatotoxic products. Moreover, Kupffer cells in coculture with hepatocytes did not augment hepatocyte injury after hypoxia-reoxygenation. Likewise, in cocultures of Kupffer cells and SECs, the presence of the Kupffer cell failed to enhance endothelial injury following hypoxia-reoxygenation, and these cocultures did not produce superoxide after reoxygenation. Thus, despite other evidence that Kupffer cells are activated in the intact liver during reperfusion injury, when present in isolation, only endothelial cells possess the innate capacity to undergo hypoxia-reoxygenation injury. Furthermore, changes in oxygen tension alone are not sufficient to activate Kupffer cells to secrete superoxides or other cell products that are toxic to hepatocytes or endothelial cells. It is concluded that SECs play a central role in hypoxia-reoxygenation injury, and the factors that activate Kupffer cells in vivo require further study.